Tolerance induction is likely to be essential to the success of clinical xenotransplantation. Studies in pig-to- mouse models, including one in which human T cells develop in porcine thymus grafts, have demonstrated that thymic xenografts induce robust donor-specific tolerance. Thymic transplantation has also been shown to prolong porcine organ survival in nonhuman primates. The overall objective of this proposal is to optimize porcine thymic transplantation as a means of xenograft tolerance induction. Our specific hypotheses for this project are: 1) a recipient conditioning regimen that can prevent initial thymic xenograft rejection without damaging the graft function or disturbing T cell "education" in the graft is required for tolerance induction through thymic xenotransplantation; 2) when tolerance is achieved by thymic xenotransplantation, the ability of porcine thymic grafts to eliminate "self (human)-reactive T cells (especially those reacting to peripheral tissue antigens) and to support the development of human regulatory T cells will be of importance for self tolerance; and 3) the long-term immunocompetence of the recipients will depend on the ability of human T cells developing in porcine thymus to interact with human ARC in the periphery. As detailed in the Preliminary Studies section, NOD/SCID mice reconstituted with a functional human immune system can mediate strong immune responses against porcine xenoantigens and reject porcine xenografts in vivo. We will use this mouse mode to address the above hypotheses. In Aim 1, we will identify the important effector cell populations in the rejection of porcine thymic xenografts in humanized NOD/SCID mice, and determine the extent of human immune tolerance to porcine xenografts achieved through thymus transplantation. In Aim 2, we will determine the effects of immunosuppressive drugs on human thymopoiesis in porcine thymic grafts and on tolerance induction by porcine thymus transplantation. In Aim 3 we will assess and improve the survival, function, and regulatory T cell activity of human T cells that develop in xenogeneic pig thymus. In Aim 4, we plan to compare the strength of human and pig MHC-restricted immune responses of human T cells developing in porcine versus human thymus grafts. These studies will have direct implications for the primate studies in Projects 1 and 2, and along with other projects of this PPG, ultimately provide a novel approach for tolerance induction in clinical xenotranspiantation.